reaction mix  (TaKaRa)

Journal: bioRxiv

Article Title: Optimizing in vitro Transcribed CRISPR-Cas9 Single-Guide RNA Libraries for Improved Uniformity and Affordability

doi: 10.1101/2025.03.24.644170

Figure Lengend Snippet: a. We PCR-amplified microarray-derived 98-nt oligo subpools, each containing spacers corresponding to individual sgRNA libraries. Each oligo was designed with forward and reverse PCR primer sites, a T7 promoter followed by 1-2 guanines, unique 20 nucleotide spacer sequences, and a BsaI type IIs restriction site. Golden Gate Assembly (GGA) was used to add the conserved sgRNA scaffold sequence to the oligos. The resulting GGA products were used as templates for pooled IVT of sgRNA libraries using T7 RNAP. b. To evaluate the initial performance of our sgRNA synthesis workflow, we manually pooled 18 column-synthesized oligos with 18 unique spacer sequences, converting them to dsDNA by extending the reverse primer, followed by GGA and IVT. Successful synthesis of pooled and individual sgRNAs (100 bases) was confirmed by 10% TBE-urea denaturing electrophoresis, followed by SYBR-Gold post-staining. c. Ten sgRNA libraries containing 206 to 2,626 spacers were synthesized from microarray-derived (array) oligos. The percent coverage of the microarray-derived libraries (orange dots) was compared to the percent coverage of the 18-plex sgRNA library (teal dot) with column derived oligos. We find an inverse correlation between coverage and scale (Pearson correlation analysis, r = −0.58). d. Comparison of spacer distribution uniformity between a microarray-derived 206-plex sgRNA library and a library of 72 independently transcribed sgRNAs from New England Biolabs (NEB). Normalized abundance is shown, with spacers ranked in descending order.

Article Snippet: First, components were added in the following order to prepare a 100 µL aqueous phase reaction: 2.5 µL of Murine RNase inhibitor (40,000 units/mL), nuclease-free water, 0.01-9 pmol (1-800 ng) of template DNA, 10 µL of 100 mM dithiothreitol, 10 µL of 10X RNA polymerase reaction buffer, 10 µL of 25 mM ribonucleotide solution mix, 10 µL of T7 RNA polymerase (50,000 units/mL), and 10 µL of 20 mg/mL recombinant albumin (NEB).

Techniques: Amplification, Microarray, Derivative Assay, Sequencing, Synthesized, Electrophoresis, Staining, Comparison

Journal: bioRxiv

Article Title: Optimizing in vitro Transcribed CRISPR-Cas9 Single-Guide RNA Libraries for Improved Uniformity and Affordability

doi: 10.1101/2025.03.24.644170

Figure Lengend Snippet: a. Log₂ fold change of observed vs. expected spacer abundance for each nucleotide at the first 10 positions of 20-nt spacers in a 389-plex sgRNA library transcribed from oPool oligos. Observed values are based on spacer frequencies from RNA-seq data, while expected values assume uniform spacer distribution. Positive log₂ fold changes (orange) indicate enrichment, negative values (blue) indicate depletion, and white indicates no change. b. Fraction of spacers containing homopolymer stretches within the first four nucleotides at the 5’ end, analyzed from the same RNA-seq dataset as in a. c. Log₂ fold change of spacer abundance as in A, but for 389 spacers with a 5’ guanine tetramer added at positions +1 to +4 downstream of the T7 promoter. Nucleotide positions 1-10 along the spacer are immediately downstream of the 5′ tetramer. The same color scale applies as in A. d. Normalized fraction of spacer reads relative to total reads for 389 spacers with a single guanine at the first spacer position (solid lines, n = 3) versus 389 spacers padded with 5′ guanine tetramers (dashed lines, n = 3). Spacers are ranked by descending abundance. Each library, transcribed in vitro from 400 ng of template DNA in a 20 μL reaction volume, is represented by a distinct color. Rank-order curves with listed Gini Coefficients (Gini) quantify the inequality in spacer representation across library types. e. Gini Coefficients for 135 bp DNA libraries (coral) containing 389 spacers and their corresponding sgRNA libraries (teal), in vitro transcribed by T7 RNAP using 100 ng of input DNA in a 100 µL reaction. DNA libraries were generated from microarray-derived oligos with spacers starting with a 5’ guanine (left) or oPool-derived oligos with spacers padded with a 5’ guanine tetramer (right). Spacer distributions for each library format are shown as in d .

Article Snippet: First, components were added in the following order to prepare a 100 µL aqueous phase reaction: 2.5 µL of Murine RNase inhibitor (40,000 units/mL), nuclease-free water, 0.01-9 pmol (1-800 ng) of template DNA, 10 µL of 100 mM dithiothreitol, 10 µL of 10X RNA polymerase reaction buffer, 10 µL of 25 mM ribonucleotide solution mix, 10 µL of T7 RNA polymerase (50,000 units/mL), and 10 µL of 20 mg/mL recombinant albumin (NEB).

Techniques: RNA Sequencing, In Vitro, Generated, Microarray, Derivative Assay